Preparation method and application of suvotinib standard substance in human serum

Abstract

The invention relates to a preparation method and application of a suvortinib standard substance in human serum, and belongs to the field of medical examination. The standard substance is characterized by comprising the following three levels of suvaltinib solutions: a level 1: 10.00-50.00 [mu] g / L, a level 2: 10.00-50.00 [mu] g / L, and a level 3: 10.00-50.00 [mu] g / L; the level 2 is 450.00 to 750.00 [mu] g / L; the level is 3: 1200.00 to 2000.00 [mu] g / L; wherein the level 1 corresponds to the concentration range of the blood concentration trough of the suvortinib in the serum of a patient taking the medicine; the level 2 is an effective treatment blood concentration range; and the level 3 is the peak concentration range of the blood concentration. The standard substance can be used as a conventional detection limit and a reportable range of suvortinib in human serum. The standard substance has good uniformity, stability and interoperability, and can be applied to the fields of accuracy evaluation of blood concentration detection, detection result value traceability, performance evaluation of medical equipment, instrument calibration, research and development of medical equipment and the like.

Description

Technical Field

[0001] This application belongs to the field of medical testing technology, specifically relating to a method for preparing suvortinib standard material in human serum and its application. Background Technology

[0002] Suvorinib, a breakthrough therapy for EGFR exon 20 insertion mutation (20ins) non-small cell lung cancer (NSCLC), has successfully filled the treatment gap for this refractory lung cancer subtype, marking a significant milestone for innovative Chinese drugs in the field of targeted lung cancer therapy. Currently, the drug is approved for clinical use under conditional approval, requiring continuous monitoring of its efficacy and safety to ensure its real-world application meets expectations. Precise monitoring of blood drug concentrations is a core component of this monitoring system.

[0003] Currently, the analysis of suvortinib blood concentrations faces significant technical challenges: the dual lack of standardized detection methods and traceable reference materials leads to significant differences in test results between different laboratories. Existing mainstream detection methods, such as high-performance liquid chromatography (HPLC), are limited by sensitivity and detection limits, making accurate quantification difficult; while theoretically superior HPLC-tandem mass spectrometry (HPLC-MS / MS) lacks standardized detection methods. Notably, the weak polarity of suvortinib makes the HPLC separation and detection system developed by the China National Institutes for Food and Drug Control (NIFDC) for highly polar tyrosine kinase inhibitors unsuitable for direct application. Furthermore, pure reference materials and human serum matrix reference materials for this drug are currently unavailable both domestically and internationally. As a small molecule compound, it is susceptible to environmental factors such as light, heat, humidity, and oxygen during storage and use, exhibiting significant stability issues, and relevant solutions are rarely reported in existing literature.

[0004] Establishing a precise blood drug concentration monitoring system, developing standardized reference measurement methods, and researching national standard substances are key paths to overcoming the current predicament. This initiative will not only meet the requirements for continuous evaluation of drug safety and efficacy after conditional marketing authorization, but also improve the consistency of results between laboratories through unified testing standards, providing a reliable basis for individualized clinical treatment.

[0005] Establishing a scientific, stable, reproducible, and internationally aligned standard substance preparation system is of strategic significance for promoting the standardization and international recognition of clinical testing. As a representative domestically developed innovative targeted drug, the successful development of suvortinib standard substances will lay a standardized foundation for its blood drug concentration testing, helping my country build an independent and controllable quality standard system for innovative drugs and enhancing China's international influence in the field of lung cancer targeted therapy. Summary of the Invention

[0006] This invention provides a method for detecting suvortinib candidate standard substances in human serum using liquid chromatography-mass spectrometry (LC-MS). The standard substance undergoes systematic homogeneity testing, stability studies, and uncertainty assessment. Experimental results show that the suvortinib candidate standard substance prepared by this invention meets the technical requirements for clinical application standard substances. This method fills the gap in suvortinib standard substances and detection methodologies in human serum both domestically and internationally, and lays the foundation for the standardization of suvortinib blood concentration detection in subsequent clinical practice.

[0007] The specific solution provided by this invention is as follows:

[0008] The first aspect of this invention discloses a method for preparing a suvortinib standard substance in human serum, characterized in that the suvortinib standard substance in human serum comprises suvortinib solutions at the following three concentration levels:

[0009] Level 1: 10.00~50.00μg / L;

[0010] Level 2: 450.00~750.00μg / L;

[0011] Level 3: 1200.00~2000.00μg / L;

[0012] Level 1 represents the trough concentration range of suvortinib in the serum of patients taking the drug; Level 2 represents the effective therapeutic concentration range; and Level 3 represents the peak concentration range, which can be used as the routine detection limit and reportable range of suvortinib in human serum.

[0013] The preparation method includes the following steps:

[0014] Step 1. Design of the protection buffer solution

[0015] The chemical structure and instability mechanism of suvortinib were analyzed. Based on the design concepts of anti-oxidation, prevention of optical degradation, maintenance of structural stability and anti-corrosion treatment, its protective buffer system was designed.

[0016] Step 2: Preparation of Protective Buffer Fluid

[0017] Based on the design principles of anti-oxidation, prevention of optical degradation, maintenance of structural stability and anti-corrosion treatment, suitable chemical raw materials were screened, and protective buffer solutions for suvortinib standard substances were prepared with the optimization criteria of maintaining stability, ensuring homogeneity and interoperability.

[0018] Step 3. Preparation of mother liquor:

[0019] Accurately weigh the suvortinib raw material and dissolve it in a standard substance-specific protective buffer solution to prepare three different concentrations of suvortinib stock solutions. The concentration of each stock solution is 20 times that of the corresponding three target concentration levels.

[0020] Step 4. Preparation of standard substances:

[0021] Add the suvortinib stock solution obtained in step 3 to the human serum solution at a volume ratio of 1:19, mix thoroughly, dispense into 50.0 μL / bottle, cap and seal, and store in an ultra-low temperature environment of -70℃.

[0022] Step 5. Homogeneity analysis:

[0023] Sampling and testing of suvortinib standard material in aliquoted and stored human serum were conducted, and all test data were recorded completely. Homogeneity analysis and evaluation were performed based on the test results. The criteria for determining acceptable homogeneity are as follows:

[0024] (1) The coefficient of variation of precision within the bottle must be ≤2.0%;

[0025] (2) The coefficient of variation of precision between bottles must be ≤3.0%;

[0026] Step 6. Stability Analysis:

[0027] Periodic sampling and testing of suvortinib standard material in aliquoted and stored human serum were conducted, and all test data were systematically recorded. Based on the measurement data, the stability of the standard material was comprehensively analyzed and evaluated. The stability standards were as follows: at an ultra-low temperature of -70℃, the shelf life can reach more than one year; at a low temperature of -20℃, it can be stably stored for more than six months; at a low temperature of 2-8℃, it can be stably stored for more than five days.

[0028] Step 7. Assignment and Uncertainty Assessment:

[0029] Multiple samples were taken and tested from the aliquoted and stored human serum containing suvortinib reference material. All test data were recorded in detail. The suvortinib reference material was then assigned a value, which included mean determination and uncertainty assessment.

[0030] Preferably, in the preparation method, the protective buffer solution of the standard substance is prepared by dissolving 0.4-0.6 g / L of antioxidant, 0.6-0.9 g / L of preservative, and 60-70 g / L of cryoprotectant in commercial human serum at a mass-volume ratio, and adjusting the pH value to 7.0-7.5;

[0031] Among them, the antioxidant is selected from one or more of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG);

[0032] Preservatives: selected from one or more of isobutylparaben, isopropylparaben, propylparaben, and paraben;

[0033] Cryoprotectant: Selected from one or more of ethylene glycol, sucrose, trehalose, and hydroxyethyl starch.

[0034] More preferably, the protective buffer solution comprises: 0.5 g / L butylated hydroxyanisole (antioxidant), 0.8 g / L isobutyl p-hydroxybenzoate (preservative), and 65 g / L hydroxyethyl starch (cryoprotectant).

[0035] Preferably, the detection in steps 3 to 5 is performed using liquid chromatography (LC) or liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0036] Preferably, the operating conditions for the liquid chromatography-tandem mass spectrometry method are as follows:

[0037] (1) Gefitinib national standard material pure product was selected as internal control;

[0038] (2) Chromatographic column: inner diameter 2.1 mm, length 50 mm, particle size 4.0 μm, average pore size The C18 reversed-phase chromatographic column.

[0039] (3) Flow rate: 0.1-0.2 mL / min;

[0040] (4) Mobile phase gradient: 0.1% formic acid acetonitrile solution (1-3 min); 100% acetonitrile solution (4-8 min); 0.1% formic acid solution (8-10 min).

[0041] (5) Mass spectrometry operating conditions: voltage 4.5-5.5KW, curtain gas is nitrogen, gas flow rate 5.0-8.0L / min.

[0042] Preferably, the concentration of the suvortinib standard substance in the human serum at a coverage factor k = 2 is defined as follows:

[0043] Level 1: 43.05 ± 0.64 μg / L

[0044] Level 2: 603.71±8.27 μg / L

[0045] Level 3: 1509.14±21.34μg / L.

[0046] More preferably, in the preparation method described above, the pretreatment of the suvortinib standard substance in the human serum taken before detection includes: adding 4.0 μL of suvortinib standard substance sample in human serum to 2.0 mL of acetonitrile, vortexing and mixing for 5-10 min, allowing to stand and precipitate for 30-60 min, centrifuging at 4000 g for 10 min, and taking the sample supernatant for detection.

[0047] In a second aspect, the present invention provides a standard substance of suvortinib in human serum obtained by the above preparation method, which consists of the following three concentration levels:

[0048] Level 1: 10.00~50.00μg / L;

[0049] Level 2: 450.00~750.00μg / L;

[0050] Level 3: 1200.00~2000.00μg / L;

[0051] This standard reference material meets the following technical specifications:

[0052] Uniformity: Intra-bottle precision coefficient of variation ≤ 0.87%, inter-bottle precision coefficient of variation ≤ 0.14%.

[0053] Stability: It can be stably stored for more than 1 year in an ultra-low temperature environment of -70℃; it can be stably stored for more than 6 months in a low temperature environment of -20℃; and it can be stably stored for more than 5 days in a low temperature environment of 2-8℃.

[0054] In a third aspect, the present invention provides a protective buffer for suvortinib standard material, which is prepared by dissolving 0.4-0.6 g / L of antioxidant, 0.6-0.9 g / L of preservative, and 60-70 g / L of cryoprotectant in commercial human serum at mass-volume ratios, and adjusting the pH value to 7.0-7.5.

[0055] Among them, the antioxidant used is butylated hydroxyanisole at a concentration of 0.5 g / L, the preservative is isobutyl p-hydroxybenzoate at a concentration of 0.8 g / L, and the cryoprotectant is hydroxyethyl starch at a concentration of 65 g / L.

[0056] In a fourth aspect, this study developed a liquid chromatography-mass spectrometry (LC-MS) method for the detection of suvortinib standard material. This method innovatively uses structurally similar compounds as internal controls, effectively solving the problem of missing isotope internal controls. Simultaneously, the stationary phase (using a novel bonded-phase column) and mobile phase system of the detection method were constructed and optimized, further refining the flow rate, liquid composition, and time of the mobile phase. Regarding the bonding of the stationary phase (column), an octadecyl complex carboxyl porous polymer was selected.

[0057] In a fifth aspect, the present invention provides that the above-mentioned human serum suvortinib standard material can be used in any of the following aspects:

[0058] (1) Medical device research and development;

[0059] (2) Evaluation of the accuracy of blood drug concentration detection;

[0060] (3) Calibration of testing instruments

[0061] (4) Traceability of test results;

[0062] (5) Performance evaluation of medical devices and equipment.

[0063] The technical advantages of this invention are:

[0064] (1) Reliable stability: The prepared human serum suvortinib standard material effectively solved the stability problem in the solution system. Specifically, it can be stably stored for 7 days at 28℃, stably stored for 12 months at -20℃, and stored for 24 months at -70℃ ultra-low temperature. It can also be stably maintained for 7 days under 28℃ transportation conditions.

[0065] (2) Good homogeneity: The homogeneity was verified by the in-bottle precision coefficient of variation (CV) of the standard substance. 瓶内 ≤0.87%, inter-bottle precision coefficient of variation (CV) 瓶间 The homogeneity of the reference material was ≤0.14%, which was significantly better than the standard for homogeneity (CV). 瓶内 ≤2.0%, CV 瓶间 ≤3.0%.

[0066] (3) Innovative Detection Methods: Based on the working principle of liquid chromatography-mass spectrometry (LC-MS), novel detection conditions for the stationary phase (innovative bonded phase) and mobile phase were established, and a novel detection method was developed. This provides accurate, stable, and reliable technical support for the clinical detection of suvortinib blood drug concentrations.

[0067] (4) Strong matrix simulation: Human serum is used as the matrix, and the matrix composition highly simulates the actual clinical serum test sample; at the same time, a special protective buffer formula is developed to maintain its stability.

[0068] (5) Filling the technological gap at home and abroad: The standard substance of suvortinib in human serum and its blood drug concentration detection method are both the first of their kind at home and abroad. Attached Figure Description

[0069] Figure 1 This is a flowchart illustrating the standard substance of suvortinib in human serum and its preparation method as described in this invention. Detailed Implementation

[0070] The present invention will be further described below with reference to exemplary embodiments. These embodiments do not constitute a limitation on the scope of protection of the invention. Those skilled in the art should understand that appropriate modifications or substitutions can be made to the details and form of the technical solutions without departing from the core spirit and scope of protection of the present invention, and such adjustments all fall within the scope of protection of the present invention.

[0071] Example 1: Preparation of suvortinib standard material in human serum

[0072] Step 1. Preparation of Mother Liquor

[0073] 1.1 Preparation of Standard Material Protective Buffer

[0074] Commercially available human serum (purchased from the Sigma website) was taken and mixed with 0.5 g / L butylated hydroxyanisole (antioxidant), 0.8 g / L isobutyl p-hydroxybenzoate (preservative), and 65.0 g / L hydroxyethyl starch (cryoprotectant) at a mass-to-volume ratio. The pH of the solution was adjusted to 7.0–7.5. The solution was then mixed at 2–8 °C using an ultrasonic magnetic stirrer for 30 min. After filtration through a 0.45 μm filter membrane, the standard substance protection buffer was obtained.

[0075] The above formulation is the optimal formulation for the standard substance protection buffer provided by the invention.

[0076] Other formulations provided by this invention are summarized as follows:

[0077] In commercially available human serum, 0.4–0.6 g / L of antioxidant, 0.6–0.9 g / L of preservative, and 60–70 g / L of cryoprotectant are added at a mass-to-volume ratio.

[0078] The antioxidant can be selected from one or more of butylated hydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone, and propyl gallate.

[0079] The preservative is selected from one or more of isobutyl paraben, isopropyl paraben, propyl paraben, and paraben;

[0080] The cryoprotectant is selected from one or more of ethylene glycol, sucrose, trehalose, and hydroxyethyl starch.

[0081] 1.2 Preparation of Mother Liquor

[0082] Accurately weigh sulvertinib (chromatographic grade, available from Aladdin, Sigma, or Chemicalbook) at 200 times the target concentration, add it to 10.0 mL of the above standard substance protection buffer, and mix thoroughly using an ultrasonic magnetic stirrer at 2-8°C.

[0083] Step 2. Preparation of standard substances

[0084] Add the suvortinib stock solution to the human serum solution at a volume ratio of 1:19, and mix thoroughly for 30-60 minutes using an ultrasonic magnetic stirrer at 2-8℃.

[0085] Step 3. Packaging and Storage

[0086] The above solution was dispensed into 2.0 mL brown vials, 50.0 μL per vial, and sealed with caps and stored at -70°C.

[0087] Step 4. Equipment Calibration

[0088] 4.1 Preparation of calibrators

[0089] Chromatographically pure suvortinib raw material was dissolved in acetonitrile solution to prepare a series of linear calibrators with concentrations of 10 μg / L, 50 μg / L, 200 μg / L, 800 μg / L, 1200 μg / L, 1500 μg / L, and 2000 μg / L.

[0090] 4.2 Equipment Calibration

[0091] According to the equipment instruction manual, the high performance liquid chromatography-tandem mass spectrometer was calibrated using the above-mentioned calibrators.

[0092] Example 2: Establishment of a liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection method

[0093] 1. Sample pretreatment

[0094] Take 4.0 μL of human serum containing suvortinib standard material prepared in Example 1, add 2.0 mL of acetonitrile, vortex mix for 5-10 min, let stand for 30-60 min to precipitate, centrifuge at 4000 g for 10 min, and take the supernatant for subsequent detection.

[0095] 2. High-performance liquid chromatography separation

[0096] (1) Equipment and reagent information is shown in Table 1

[0097] Table 1. Equipment and Reagent Information

[0098]

[0099] (2) Separation and elution conditions: injection volume 10.0 μL, flow rate 0.30 mL / min

[0100] The mobile phase gradient elution program is as follows: 0.1% formic acid acetonitrile solution for 1-3 min, 100% acetonitrile solution for 4-8 min, and 0.1% formic acid solution for 8-10 min. For specific parameters, please refer to Table 2.

[0101] Table 2. Separation and elution conditions for high performance liquid chromatography

[0102]

[0103]

[0104] 3. Mass spectrometry detection

[0105] (I) Equipment Information: Ion trap mass spectrometer (model: Qtrap4000, AB SCIEX, USA), equipped with an electrospray ionization source (ESI), detection mode is positive ion multiple reaction monitoring (MRM); injection volume is 1.0 μL.

[0106] (II) Setting operating parameters for the mass spectrometer

[0107] Table 3. Mass Spectrometer Operating Parameters

[0108]

[0109] Example 3. Uniformity Detection

[0110] 1. Sample Sampling

[0111] For each level of standard material, 15 bottles were randomly selected from the total sample (total sample > 400 bottles) and numbered sequentially.

[0112] 2. Sample testing

[0113] The LC-MS / MS method established in Example 2 was used to test each sample three times.

[0114] The test sequence is as follows: 1, 3, 5, 7, 9, 11, 13, 15, 2, 4, 6, 8, 10, 12, 14, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 12, 14, 1, 3, 5, 7, 9, 11, 13, 15, with test data recorded simultaneously.

[0115] 3. Statistical Analysis

[0116] The results were analyzed using one-way ANOVA using iMAST software.

[0117] The test data were statistically analyzed using the following formula:

[0118]

[0119]

[0120] ss 瓶内 =ss 总和 -ss 瓶间 ;

[0121]

[0122] v1 = α-1;

[0123] v2 = N - α;

[0124]

[0125] In the formula:

[0126] SS = variance, x i = Specifies the i-th measurement value or calculation result of the parameter. n i = Number of repeated measurements for sample i, x ij = Sample i, j-th measurement value, MS = mean square, v = degrees of freedom, F represents the F-test value, n0 = number of valid tests, α = number of samples drawn, N = total number of tests, S bb = Inter-bottle standard deviation; s r =Intra-bottle standard deviation; when the statistical result F≤10, the homogeneity between standard bottles is considered good, and it can be used as a standard. The average of all test results is calculated, and then cv is performed. 瓶间 and CV 瓶内 Calculation;

[0127] When the statistical result F>10, the homogeneity between standard bottles is considered poor, and they are not suitable as standard substances; therefore, cv is no longer calculated. 瓶间 and CV 瓶内 ;

[0128] When the statistical result F≤1, the within-bottle standard deviation is used instead of the between-bottle standard deviation, i.e., S. bb =S r .

[0129] 4. Results

[0130] The results of the homogeneity assessment of the standard materials are as follows:

[0131] Level 1: F = 0.61; cv 瓶间 =0.87%; cv 瓶内 =0.87%;

[0132] Level 2: F = 3.14; cv 瓶间 =0.11%; cv 瓶内 =0.13%

[0133] Level 3: F = 1.09; cv 瓶间 =0.14%; cv 瓶内 =0.82%

[0134] The above test results prove that the suvortinib standard in the human serum prepared by this invention has good homogeneity and meets the technical requirements for homogeneity of standard substances.

[0135] Example 4. Stability Testing

[0136] 1. Experimental Design: The specific design for the stability test is shown in Table 4.

[0137] Table 4. Stability Testing Design

[0138] Stability classification Time settings Stability at 2℃-8℃ Days 5, 4, 3, 2, and 1 before testing Bottle opening stability at 2℃-8℃ Days 5, 4, 3, 2, and 1 before testing Melting stability at 2℃-8℃ Days 5, 4, 3, 2, and 1 before testing Stability at room temperature (20℃-30℃) 24 hours, 12 hours, 6 hours, 3 hours and 1 hour before testing Stability during transport at 2℃~8℃ Days 5, 4, 3, 2, and 1 before testing Long-term stability at -20℃ 1 month, 2 months, 3 months, 5 months, 9 months and 12 months Long-term stability at -70℃ 1 month, 3 months, 5 months, 12 months, 18 months and 24 months

[0139] 2. Stability test at 2℃-8℃:

[0140] Six vials of suvortinib standard material from three levels of human serum were randomly selected from the total sample. On days 5, 4, 3, 2 and 1 before the test, the samples were placed at 2℃-8℃. On day 0, the standard material was repeatedly tested three times using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0141] 3. Bottle-opening stability test at 2℃-8℃:

[0142] Six vials of suvortinib standard material from three levels of human serum were randomly selected from the total sample. The samples were opened at 2℃-8℃ on days 5, 4, 3, 2 and 1 before the test. On day 0, the standard material was tested three times using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0143] 4.2℃-8℃ Remelting Stability Test:

[0144] Six vials of suvortinib standard material from three levels of human serum were randomly selected from the total sample. The standard materials were thawed in a refrigerator at 2℃-8℃ on days 5, 4, 3, 2, 1 before the test and on day 0. On day 0, the standard materials were tested three times using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0145] 5.20℃-30℃ room temperature stability test:

[0146] Five bottles of suvortinib standard material from three levels of human serum were randomly selected from the total sample. The samples were placed at room temperature of 20℃-30℃ at 24 hours, 12 hours, 6 hours, 3 hours and 1 hour before the test. The standard material was repeatedly tested three times at hour 0 using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0147] Stability test during transportation at 6.2℃~8℃:

[0148] Six vials of suvortinib standard material from three levels of human serum were randomly selected from the total sample and transported at 2℃-8℃. On days 1, 2, 3, 4 and 5 after transportation, the standard material was repeatedly detected three times using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0149] 7. Long-term stability test at -20℃:

[0150] Six bottles of suvortinib standard material from three levels of human serum were randomly selected from the total sample and placed in a -20°C freezer. At the 1st, 2nd, 3rd, 5th, 9th and 12th month after storage, the standard material was repeatedly detected three times using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0151] 8. Long-term stability test at -70℃:

[0152] Six bottles of suvortinib standard material from three levels of human serum were randomly selected from the total sample and placed in a -70°C freezer. At 1 month, 3 months, 5 months, 12 months, 18 months and 24 months after storage, the standard material was repeatedly detected three times using the LC-MS / MS method established in Example 2. The data were recorded and statistically analyzed.

[0153] 9. Statistical Analysis:

[0154] The results were analyzed using linear regression analysis with iMAST software, and the mean detection results are shown in Table 5 below.

[0155] Table 5 Stability test results

[0156]

[0157] The above experiments show that each treatment satisfies |β1|≤t 0,95,n-2x s(β1) indicates that the human serum suvortinib standard material prepared in this invention has good stability under various conditions such as long-term storage, opening, remelting, short-term storage and transportation, and meets the stability qualification standard for standard materials.

[0158] Example 5. Standard reference value determination

[0159] 1. Standard reference mean value detection

[0160] Fifty vials of suvortinib standard material from each of the three levels of human serum were randomly selected from the total sample. Each standard material was analyzed once using LC-MS / MS. Ten vials were tested daily for five consecutive days. All test results were recorded, and the population mean and standard deviation were calculated. The mean test results are shown in Tables 6-8 below:

[0161] Table 6. Detection data of suvortinib standard substance in human serum at level 1, and overall mean and standard deviation.

[0162]

[0163] Table 7. Detection data of suvortinib standard substance in human serum at level 2, and overall mean and standard deviation.

[0164]

[0165] Table 8. Detection data of suvortinib standard substance in human serum at level 3, and overall mean and standard deviation.

[0166]

[0167] 2. Uncertainty Assessment of Standard Reference Materials

[0168] 2.1 Analysis of Uncertainty Sources

[0169] According to the "Evaluation of Measurement Uncertainty of Standard Reference Materials for In Vitro Diagnostic Reagents" (YY / T 1709-2020), the uncertainties introduced by the suvortinib standard reference material in human serum include: value correlation (measurement precision, working standard reference material), homogeneity, and stability (open bottle stability, remelting stability, and shelf-life stability).

[0170] 2.2 Sampling of Standard Reference Materials

[0171] 100 vials / level of suvortinib standard substances (level 1, level 2, level 3) were randomly selected from human serum.

[0172] 2.3 Measurement Procedure

[0173] The LC-MS / MS method and related conditions determined in Example 2 were used.

[0174] 2.4 Uncertainty Assessment Method

[0175] (1) Measurement precision

[0176] Using the above measurement procedure, suvortinib standard substances (level 1, level 2, and level 3) in 50 randomly selected bottles / levels of human serum were tested: 10 bottles were tested daily, with each bottle tested three times, for 5 consecutive days. The test results were recorded, and the uncertainty of the data was analyzed using the following formula:

[0177]

[0178] u rep.rel = Measurement precision uncertainty;

[0179] n = the number of repeated measurements;

[0180] The results are as follows:

[0181] Table 9. Uncertainty introduced by the precision measurement of suvortinib standard material in human serum at level 1

[0182]

[0183]

[0184] Table 10. Uncertainty introduced by the precision measurement of suvortinib standard material in level 2 human serum

[0185]

[0186] Table 11. Uncertainty introduced by the precision measurement of suvortinib standard material in level 3 human serum

[0187]

[0188]

[0189] (2) Uncertainty introduced by working standard materials

[0190] Consult the certificate of the working reference material. The uncertainty introduced by the reference material is calculated using the following formula:

[0191]

[0192] in,

[0193] C wcal = Standard value of suvortinib; u wcal = Expanded uncertainty of a constant value; k is the coverage factor, which equals 2. Calculation results:

[0194] Table 12. Uncertainty introduced by working standard materials

[0195]

[0196] (3) Uniformity

[0197] Using the above measurement procedure, the concentration of suvortinib standard material (level 1 and level 2) in 15 randomly selected and numbered bottles / levels of human serum was tested; the test order was: 1, 3, 5, 7, 9, 11, 13, 15, 2, 4, 6, 8, 10, 12, 14, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 2, 4, 6, 8, 10, 12, 14, 1, 3, 5, 7, 9, 11, 13, 15; the data results were recorded, and uncertainty analysis was performed using the following formula:

[0198]

[0199] in:

[0200] n = the number of repeated tests for bottle i; MSbb =Mean square between bottles; MS wb =Square hull inside the bottle; u bb = Uncertainty of homogeneity.

[0201] The calculation results are shown in Tables 13-15.

[0202]

[0203]

[0204]

[0205] (4) Stability after opening

[0206] Twelve vials / level of human serum containing suvortinib standard material were randomly selected. Two vials of standard material were opened and placed in a refrigerator at 2℃~8℃ on days 5, 4, 3, 2, and 1 before testing, and on day 0. On day 0, the above measurement procedure was repeated three times for each vial of standard material. The test data were recorded, and the uncertainty was calculated using the following formula:

[0207]

[0208] u st1 = t × s(b1);

[0209] In the formula:

[0210] u st1 = Uncertainty introduced by opening the bottle; t = Number of time points; s(b1) = Uncertainty of the slope;

[0211] b1 = slope; b0 = intercept; s = standard deviation of the line; x i = point in time;

[0212]

[0213] The data and calculation results are shown in Table 16:

[0214] Table 16. Uncertainties introduced by bottle-opening stability

[0215]

[0216]

[0217] (5) Remelting stability

[0218] Twelve vials / level of human serum containing suvortinib standard material were randomly selected. Two vials of standard material were taken daily on days 5, 4, 3, 2, and 1 before testing, and on day 0. The samples were then reconstituted and stored at 2℃–8℃. On day 0, the above measurement procedure was repeated three times for each vial of standard material. The test data were recorded, and the uncertainty was calculated using the following formula:

[0219]

[0220] u st2 = t × s(b1),

[0221] In the formula:

[0222] u st2 = Uncertainty introduced by complex fusion; t = Number of time points; s(b1) = Uncertainty of the slope; b1 = Slope;

[0223] b0 = intercept; s = standard deviation of the line; x i = point in time;

[0224]

[0225] Test and calculation results:

[0226] Table 17. Uncertainties introduced by the stability of fusion

[0227]

[0228] (6) Shelf life stability:

[0229] It has been verified that the uncertainty introduced by the expiration date is less than 1 / 3 of the maximum uncertainty, and therefore is not included in the total uncertainty.

[0230] (7) Uncertainty introduced by the constant value

[0231]

[0232] in:

[0233] u char.rel = Relative uncertainty of a constant; u rep.rel This represents the precision uncertainty, the values ​​of which are recorded in Table 9-11 above; u wcal.rel The value of the working standard material is shown in Table 12.

[0234] Test and calculation results:

[0235] Table 18. Uncertainty introduced by the level 1 constant value

[0236] <![CDATA[Measurement precision uncertainty u rep.rel > <![CDATA[Uncertainty u of working reference material wcal.rel > <![CDATA[Fixed relative uncertainty u char.rel > 0.000506175 0.003009027 0.003051304

[0237] Table 19. Uncertainty introduced by the level 2 constant value

[0238] <![CDATA[Measurement precision uncertainty u rep.rel > <![CDATA[Uncertainty u of working reference material wcal.rel > <![CDATA[Fixed relative uncertainty u char.rel > 0.000163887 0.003009027 0.003013487

[0239] Table 20. Uncertainty introduced by the level 3 constant value

[0240] <![CDATA[Measurement precision uncertainty u rep.rel > <![CDATA[Uncertainty u of working reference material wcal.rel > <![CDATA[Fixed relative uncertainty u char.rel > 0.000279696 0.003009027 0.003021998

[0241] (8) Uncertainty Combination

[0242] (I) Combined standard uncertainty

[0243]

[0244] u c =u c.rel ·×C;

[0245] in:

[0246] u c Indicates the combined standard uncertainty; u c.rel Indicates the composite standard relative uncertainty;

[0247] u char u represents the standard uncertainty introduced by a constant value. char =u char.rel ×C;

[0248] C represents the assigned value of the suvortinib standard substance in human serum of the product, which is the overall mean value of each level recorded in Table 6-8;

[0249] u bb The uncertainty representing homogeneity is recorded in Tables 13-15; u st1 This indicates the uncertainty introduced by opening the bottle;

[0250] u st2 This represents the uncertainty introduced by refining.

[0251] (II) Combined Standard Expanded Uncertainty

[0252] U = u c ×k;

[0253] Where k is the coverage factor (k=2)

[0254] Relevant data and calculation results are shown in Tables 21-26.

[0255] Table 21. Combined Standard Uncertainty at Level 1

[0256] Combined standard relative uncertainty Target value Combined standard uncertainty 0.007452879 43.05 0.320846441

[0257] Table 22. Combined Standard Expanded Uncertainty at Level 1

[0258] Combined standard uncertainty Inclusion factor Target value Expanded uncertainty U 0.320846441 2 43.05 0.64

[0259] Table 23. Combined Standard Uncertainty at Level 2

[0260] Combined standard relative uncertainty Target value Combined standard uncertainty 0.00685256 603.71 4.136958867

[0261] Table 24 Level 2 Combined Standard Expanded Uncertainty

[0262] Combined standard uncertainty Inclusion factor Target value Expanded uncertainty U 4.136958867 2 603.71 8.27

[0263] Table 25. Combined Standard Uncertainty at Level 3

[0264] Combined standard relative uncertainty Target value Combined standard uncertainty 0.007069339 1509.14 10.66862172

[0265] Table 26. Combined Standard Expanded Uncertainty at Level 3

[0266] Combined standard uncertainty Inclusion factor Target value Expanded uncertainty U 10.66862172 2 1509.14 21.34

[0267] (9) Determination of suvortinib standard substance in human serum

[0268] Level 1: 43.05 ± 0.64 μg / L, k = 2;

[0269] Level 2: 603.71 ± 8.27 μg / L, k = 2;

[0270] Level 3: 1509.14±21.34μg / L, k=2.

[0271] The above experiments demonstrate that the uncertainty of the suvortinib standard substance in human serum prepared by this invention is small and less than the allowable deviation range (±10.0%), meeting the qualification requirements for standard substances.

[0272] Example 6: Stability Comparison of Different Protective Buffer Systems

[0273] 1. Recipe:

[0274] The different formulations of the protective buffer system are shown in Table 27 below.

[0275] Table 27. Formulation of Protective Buffer System

[0276]

[0277] 2. Steps:

[0278] (1) Preparation of standard substances:

[0279] Suvortinib raw material was dissolved in protective buffer systems with different formulations to prepare suvortinib standard substances (concentration of approximately 400 μg / L).

[0280] (2) Constant value:

[0281] Using the established reference measurement procedure, the prepared standard substances were assigned values, and the results are shown in Table 28 below.

[0282] Table 28. Fixed Value Results

[0283] Formula 1 Formula 2 Formula 3 403.88±5.20μg / L, k=2 406.10±4.40μg / L, k=2 404.10±5.23 μg / L, k=2

[0284] (3) Refrigerated storage:

[0285] The prepared standard substances were stored in the dark at 2–8°C for one week.

[0286] (4) Detection:

[0287] Using the established reference measurement procedure, the standard substances stored under the above conditions were tested, and the test results are shown in Table 29 below.

[0288] 3. Test Results:

[0289] Table 29. Test Results

[0290] Formula 1 Formula 2 Formula 3 368.88±9.20μg / L, k=2 405.10±3.56μg / L, k=2 305.10±8.23μg / L, k=2

[0291] 4. Conclusion:

[0292] Among the above test results, the target value change of Formula 2 was the smallest, which proves that the stability protection result of the protective buffer system of Formula 2 is the best.

[0293] The technical means disclosed in this invention are not limited to the above-mentioned contents, but also include technical solutions composed of any combination of the above technical features.

[0294] The above description describes specific embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a standard substance of suvratoxini in human serum, characterized by, The human serum suvratoitin standard substance contains the following three levels: Level 1: 10.00-50.00 μg / L; Level 2: 450.00-750.00 μg / L; Level 3: 1200.00-2000.00 μg / L; Wherein, level 1 corresponds to the concentration range of trough concentration of suvratoitin in serum of patients taking medicine; level 2 is the effective therapeutic concentration range; level 3 is the peak concentration range. The standard substance can be used as the routine detection limit and reportable range of suvratoitin in human serum; The preparation method comprises the following steps: Step 1. Design of protection buffer Based on the design principles of anti-oxidation, prevention of optical degradation, maintenance of structural stability and preservation treatment, the protection buffer system of suvratoitin is designed according to the chemical structure and instability principle of suvratoitin; Step 2. Preparation of protection buffer Based on the design principles of anti-oxidation, prevention of optical degradation, maintenance of structural stability and preservation treatment, the protection buffer of suvratoitin standard substance is prepared by screening suitable chemical raw materials and taking maintenance of stability, ensuring uniformity and interoperability as the optimization standard; Step 3. Preparation of mother liquor: The suvratoitin raw material is accurately weighed, and then completely dissolved in the protection buffer of the standard substance. Through accurate configuration operation, three different concentrations of suvratoitin mother liquor are prepared, and the concentration of each mother liquor is 20 times of the target concentration level respectively; Step 4. Preparation of standard substance: The suvratoitin mother liquor is measured, and then added into the human serum solution according to the volume ratio of 1:

19. An appropriate mixing method is used to ensure uniform mixing. Then, the mixed solution is divided into 50.0 μL per bottle. After the division, the bottle is sealed in time, and then stored in an ultra-low temperature environment of-70℃; Step 5. Uniformity analysis: The suvratoitin standard substance in human serum is sampled and accurately detected, and all the detection data are recorded in detail. Based on the measured data, the uniformity of the standard substance is analyzed and evaluated. The uniformity qualified standard is as follows: (1) The in-bottle precision variation coefficient is less than or equal to 2.0%; (2) The inter-bottle precision variation coefficient is less than or equal to 3.0%; Step 6. Stability analysis: The suvratoitin standard substance in human serum is sampled and detected, and all the detection data are recorded in detail. Based on the measured data, the stability of the standard substance is analyzed and evaluated. The stability qualified standard is that the standard substance can be stably stored for more than one year in an ultra-low temperature environment of-70℃, for more than six months in a low temperature environment of-20℃, and for more than five days in a low temperature environment of 2-8℃; Step 7. Value determination The suvratoitin standard substance in human serum is sampled and detected, and all the detection data are recorded. The suvratoitin standard substance is valued, and the value determination process includes mean value determination and uncertainty evaluation.

2. The preparation method according to claim 1, wherein the protective buffer of the standard substance of sorafenib is prepared by dissolving 0.4-0.6 g / L of an antioxidant, 0.6-0.9 g / L of a preservative and 60-70 g / L of a cryoprotectant in the commercial human serum in a mass-volume ratio, and adjusting the pH value to 7.0-7.

5. wherein: The antioxidant is selected from one or more of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butylhydroquinone (TBHQ) and propyl gallate (PG). The preservative is selected from one or more of isobutyl p-hydroxybenzoate, isopropyl p-hydroxybenzoate, propyl p-hydroxybenzoate and p-hydroxybenzoic acid. The cryoprotectant is selected from one or more of ethylene glycol, sucrose, trehalose and hydroxyethyl starch.

3. The production method according to claim 1, characterized by, The protective buffer of the standard substance of sorafenib is prepared by dissolving 0.5 g / L of butylated hydroxyanisole, 0.8 g / L of isobutyl p-hydroxybenzoate and 65 g / L of hydroxyethyl starch in the commercial human serum in a mass-volume ratio.

4. The production method according to claim 1 and 2, characterized by, Before detection, the sample of the standard substance of sorafenib in the human serum is pretreated: 4.0 μL of the sample of the standard substance of sorafenib in the human serum is taken, 2.0 mL of acetonitrile is added, vortexed at a speed of 1500-2000 rpm for 5-10 min, then left to stand at room temperature for 30-60 min, and centrifuged at a centrifugal force of 4000 g for 10 min, and the supernatant is taken as the detection sample.

5. The preparation method according to claim 1, characterized in that, In steps 5-7, the detection method is liquid chromatography-tandem mass spectrometry, and the specific working conditions are as follows: (1) The pure product of the national standard substance of gefitinib is selected as the internal reference; (2) Column: C18 reversed-phase chromatographic column (porous high polymer packing, bonded phase of octadecyl complex carboxyl) with inner diameter of 2.1 mm, length of 50 mm, particle size of 4.0 μm, and average pore diameter of 10 nm. (2) Column: C18 reversed-phase chromatographic column (porous high polymer packing, bonded phase of octadecyl complex carboxyl) with inner diameter of 2.1 mm, length of 50 mm, particle size of 4.0 μm, and average pore diameter of 10 nm. (3) Flow rate: 0.1-0.2 mL / min; (4) Mobile phase gradient: 0.1% formic acid acetonitrile solution (1-3 min); 100% acetonitrile solution (4-8 min); 0.1% formic acid solution (8-10 min). (5) Mass spectrometry working conditions: voltage 4.5-5.5 KW, curtain gas nitrogen, gas flow rate 5.0-8.0 L / min.

6. The method of claim 1, wherein the step of forming the first and second layers is performed by a method selected from the group consisting of: sputtering, evaporation, and chemical vapor deposition. The concentration of the standard substance of sorafenib in the human serum is as follows: under the condition that the coverage factor k=2 and the confidence level of the measurement result is 95%, the concentration of the standard substance of sorafenib in the human serum is as follows: Level 1: 43.05±0.64 μg / L, Level 2: 603.71±8.27 μg / L, Level 3: 1509.14±21.34 μg / L.

7. The standard substance of sorafenib in the human serum prepared by the preparation method of any one of claims 1-6, covering the following three levels: Level 1: 10.00-50.00 μg / L; Level 2: 450.00-750.00 μg / L; Level 3: 1200.00-2000.00 μg / L; The performance of the standard substance can reach the following levels: In terms of uniformity, the within-bottle precision variation coefficient is ≤0.87%, and the between-bottle precision variation coefficient is ≤0.14%. In terms of stability, it can be stable for more than 1 year in an ultra-low temperature environment of -70℃, for more than 6 months in a low temperature environment of -20℃, and for more than 5 days in a low temperature environment of 2-8℃.

8. The human serum standard substance of sorafenib as claimed in claim 7 is used in any of the following aspects: (1) research and development of medical devices; (2) accuracy evaluation of blood drug concentration detection; (3) calibration of detection instruments (4) traceability of detection results; (5) performance evaluation of medical device equipment.

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